Chemical depolarization-induced SR calcium release in triads isolated from rabbit skeletal muscle.
Chemical depolarization-induced SR calcium release in triads isolated from rabbit skeletal muscle.
复制标题
从兔骨骼肌中分离出的三联体中化学去极化诱导的 SR 钙释放。
DOI：
10.1021/bi00202a015
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Morii,M
中科院分区：
文献类型：
--
作者：
Ikemoto,N;Yano,M;el-Hayek,R;Antoniu,B;Morii,M
修订稿于 1994 年 7 月 5 日收到*摘要：通过遵循用于研究带皮纤维系统中的激发-收缩耦合的相同类型的方法，研究了富含三联体的兔子骨骼肌的重微粒体部分中的激发-Ca2+释放耦合特性。将三联体与 Mg* ATP 在含有 150 mM K+、15.0-37.2 mM Na+、150-180 mM 葡萄糖酸盐-和 150-200 pM Ca2+（引发溶液）的溶液中孵育，导致 (a) 产生 T 管膜电位，使细胞质侧呈阴性，通过电位探针的电位依赖性吸收进行评估[14C] SCN-通过三联体，以及 (b) Ca2+ 主动转运至 SR 部分。根据 Cl-- 置换 [Donaldson, S. K. B. (1985) J. Gen. Physiol 86, 501-525；Donaldson, S. K. B. (1985) J. Gen. Physiol 86, 501-525； Stephenson, E. W.(1985) J. Gen. Physiol。 86, 813-832] 和 Na+ 替代 [Lamb, GD, & Stephenson, DG (1990) J. Physiol。 423, 495-517] 用于诱导带皮纤维系统收缩的方案。通过使用 Fluo-3 作为 Ca2+ 探针，在 BAPTA-钙缓冲液存在下通过停流荧光测定法测定，任一方案的离子置换过程都会从 SR 中快速释放 Ca2+。 Ca2+释放的速率常数和幅度均随着离子取代程度的增加而增加。释放动力学中离子取代依赖性的变化与完整纤维系统中 Ca2+ 瞬变的电压依赖性变化具有惊人的相似性。通过多种药物（例如 Na+-K+ 泵阻滞剂（80 pM 地高辛）和 Na+-K+ 梯度破坏剂（10/uM 莫能菌素和 10/uM 缬氨霉素））阻断 T 管极化，可显着抑制离子置换诱导的 SR Ca2+ 释放，而对多聚赖氨酸直接刺激 SR 通道诱导的 SR Ca2+ 释放没有影响。这表明离子置换诱导的 Ca2+ 释放受到 T 管电位的控制。上述结果表明，在分离的三联体中保留了去皮（或完整）肌纤维系统中 ec 耦合的基本特征，并且三联体制备可以作为简化的生理模型，用于研究 ec 耦合的分子机制。 T-tubule1 膜电位的瞬时变化导致 SR 快速释放 Ca2+ 的机制是肌肉生理学中最重要的未解决问题之一（Endo，1977；Martonosi， 1984；施奈德，1981；Fleischer 和 Inui，1989；Rios 和 Gonzalez，1991；最近的研究已经解决了耦合过程中涉及的两个主要分子成分。 T 管二氢吡啶 (DHP) 受体的 a i 亚基起着关键作用，以下事实证明了这一点：该亚基在无法进行 ec 偶联的基因缺陷小鼠中缺失（Knudson 等，1989），而其表达可重新产生 ec 偶联和电荷运动（Tanabe 等，1988a，b，1990；Adams 等， 1990），DHP 阻止 ec 耦合和电荷运动（Rios & Brum，1987）。假定的电压感测的存在
Revised Manuscript Received July 5, 1994* abstract: Excitation-Ca2+ release coupling properties in the heavy microsomal fraction of the rabbit skeletal muscle enriched in triads were investigated by following the same type of approach used for the studies of excitation-contractioncoupling in the skinned fiber system. Incubation of the triads with Mg* ATP in a solution containing 150 mM K+, 15.0-37.2 mM Na+, 150-180 mM gluconate-, and 150-200 pM Ca2+(priming solution) led to (a) the generation of a T-tubule membrane potential making the cytoplasmic side negative, as assessed by potential-dependent uptake of the potential probe [14C] SCN-by triads, and (b) active transport of Ca2+ into the SR moiety. One volume of the primed (viz., polarized and Ca2+-loaded) triads was mixed with nine volumes of depolarizationsolution according to Cl--replacement [Donaldson, S. K. B.(1985) J. Gen. Physiol 86, 501-525; Stephenson, E. W.(1985) J. Gen. Physiol. 86, 813-832] and Na+-replacement [Lamb, GD, & Stephenson, DG (1990) J. Physiol. 423, 495-517] protocols used for the induction of contraction in skinned fiber system. The ionic replacement procedure by either protocol produced a rapid release of Ca2+ from SR as determined by stopped-flow fluorometry using fluo-3 as a Ca2+ probe in the presence of BAPTA-calcium buffer. Both the rate constant and the magnitude of Ca2+ release increased with the degree of ionic replacement. The ionic replacement-dependentchanges in the release kinetics showed a striking similarity to the voltage-dependent changes of the Ca2+ transient in the intact fiber system. Blocking of T-tubule polarization by several agents, such as the Na+-K+ pump blocker (80 pM digoxin) and the Na+-K+ gradient breaker (10/uM monensin together with 10/uM valinomycin), resulted in significant inhibition of ionicreplacement-induced SR Ca2+ release, showing no effect on SR Ca2+ release induced by direct stimulation of the SR channel by polylysine. This indicates that ionic replacement-induced Ca2+ release is under the control of T-tubule potential. The above results suggest that essential features of ec coupling in the skinned (or intact) muscle fiber system are retained in the isolated triad, and the triad preparation can serve as a simplified physiological model useful for the studies of the molecular mechanism of ec coupling.The mechanism by which transient changes in the T-tubule1 membrane potential lead to rapid Ca2+ release from the SR is one of the most important unsolved questions in muscle physiology (Endo, 1977; Martonosi, 1984; Cailleet al., 1985; Schneider, 1981; Fabiato, 1989; Fleischer & Inui, 1989; Rios & Pizzaro, 1991; Rios & Gonzalez, 1991). Recent studies have resolved two major molecular components involved in the coupling processes. The a i subunit of the dihydropyridine (DHP) receptor of the T-tubule plays a critical role, as evidenced by the following facts: this subunit is missing in the dysgenic mouse incapable of ec coupling (Knudson et al., 1989), while its expression regenerates ec coupling and charge movement (Tanabe etal., 1988a, b, 1990; Adams et al., 1990), and DHPs block ec coupling and charge movement (Rios & Brum, 1987). The presence of the putative voltage-sensing